Acid-base properties of cobalt(II)-substituted carbonic anhydrases

Bertini, Ivano; Dei, Andrea; Luchinat, Claudio; Monnanni, Roberto

doi:10.1021/ic00197a012

Cited by 31 publications

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“…Co(II)-substitution has been largely exploited as a fruitful strategy to obtain structural information about the active sites of zinc enzymes by employing the spectroscopic features of Co(II) (38). This characterization has been also applied to MBLs (16,19,39,40).…”

Section: Resultsmentioning

confidence: 99%

Mimicking natural evolution in metallo-β-lactamases through second-shell ligand mutations

Tomatis

Rasia

Segovia

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Metallo-␤-lactamases (MBLs) represent the latest generation of ␤-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most ␤-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: (i) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (ii) directed evolution is able to mimic mutations that occur in nature; (iii) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and (iv) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.Co(II)-substitution ͉ directed evolution ͉ zinc enzymes ͉ antibiotic resistance T he ␤-lactam antibiotics account for more than half of the world's antibiotic market and are one of the cornerstones of antibacterial chemotherapy (1). The increasing use of these drugs, both in the clinical setting and in animal feed, induces the development of different resistance mechanisms in pathogenic and opportunistic microorganisms (2, 3). Among them, the expression of ␤-lactamases is predominant. In the last decade, there has been growing concern about the dissemination of genes coding for metallo-␤-lactamases (MBLs) among infectious microorganisms (4-7). MBLs are zinc-dependent enzymes that hydrolyze ␤-lactams by favoring the deprotonation of a metalbound water molecule, which (as a hydroxide) is a powerful nucleophile (8-11). Much recent literature has been devoted to the elucidation of the structure and mechanism of action of . Parallel to these efforts, the significant growth of new MBL sequences has revealed an initially unforeseen molecular and structural diversity in this family of enzymes (6). This diversity encompasses changes in the coordination features of the Zn(II) ions as well as in the topology of the active site. This fact has hitherto thwarted the successful development of clinically useful inhibitors. The potential danger of MBLs is further enhanced by the broad substrate spectrum displayed by these enzymes, as compared with the better-characterized Serdependent ␤-lactamas...

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Section: Resultsmentioning

confidence: 99%

Mimicking natural evolution in metallo-β-lactamases through second-shell ligand mutations

Tomatis

Rasia

Segovia

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The p H profile of k cat / K m at steady state (Figure 2B) also closely resembles the wild type enzyme, modeled by equation 4, describing a cooperative 4-proton loss with a pK a value of 7.48 ± 0.03 (compared to 7.7 ± 0.4 for the wild-type enzyme). The pK a associated with the k cat / K m p H-rate profile should reflect the apparent pK a of the metal-bound water molecule in the metal-hydroxide mechanism common to all carbonic anhydrases; the slightly more acidic pK a value for Co-HICA is within one standard error of the wild-type enzyme, but may also reflect the increased acidity of Co(II)–bound water compared to Zn(II)-bound water [32, 52-54]. Cobalt derivatives of carbonic anhydrases in general approach the functional levels (50-200%) of zinc-bound enzymes [35, 55], our results (120% of wild-type) are consistent with other cobalt(II)-substituted zinc metalloenzymes.…”

Section: Discussionmentioning

confidence: 99%

Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: Spectral evidence for allosteric regulation by pH and bicarbonate ion

Hoffmann

Samardzic

Heever

et al. 2011

Archives of Biochemistry and Biophysics

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Cobalt(II)-substituted Haemophilus influenzae β-carbonic anhydrase (HICA) has been produced by overexpression in minimal media supplemented with CoCl2, enabling kinetic, structural, and spectroscopic characterization. Co(II)-substituted HICA (Co-HICA) has comparable catalytic activity to that of wild-type enzyme with kcat = 82 ± 19 ms-1 (120% of wild-type). The X-ray crystal structure of Co-HICA was determined to 2.5 Å resolution, and is similar to the zinc enzyme. The absorption spectrum of Co-HICA is consistent with four-coordinate geometry. pH-dependent changes in the absorption spectrum of Co-HICA, including an increase in molar absorptivity and a red shift of a 580 nm peak with decreasing pH, correlate with the pH dependence of kcat/Km. The absence of isosbestic points in the pH-dependent absorption spectra suggest that more than two absorbing species are present. The addition of bicarbonate ion at pH 8.0 triggers spectral changes in the metal coordination sphere that mimic that of lowering pH, supporting its hypothesized role as an allosteric inhibitor of HICA. Homogeneously (99 ± 1% Co) and heterogeneously (52 ± 5% Co) substituted Co-HICA have distinctly different colors and absorption spectra, suggesting that the metal ions in the active sites in the allosteric dimer of Co-HICA engage in intersubunit communication.

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“…The data indicate an equilibrium between high and low pH forms, and the spectral changes parallel changes in catalytic activity [5]. More detailed solution studies show the titration curve is complex consistent with a smaller influence of other ionizable groups near the active site [4, 8]. The visible spectrum of Co(II)-CA observed at high pH shows much stronger absorbance at 640 nm (ε ~ 300 M −1 cm −1 ) than that at low pH (ε ~ 50 M −1 cm −1 ).…”

Section: Introductionmentioning

confidence: 99%

Comparison of solution and crystal properties of Co(II)–substituted human carbonic anhydrase II

Avvaru¹,

Arenas²,

Tu³

et al. 2010

Archives of Biochemistry and Biophysics

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The visible absorption of crystals of Co(II)-substituted human carbonic anhydrase II (Co(II)-HCA II) were measured over a pH range of 6.0 to 11.0 giving an estimate of pKa 8.4 for the ionization of the metal-bound water in the crystal. This is higher by about 1.2 pKa units than the pKa near 7.2 for Co(II)-CA II in solution. This effect is attributed to a nonspecific ionic strength effect of 1.4 M citrate in the precipitant solution used in the crystal growth. A pKa of 8.3 for the aqueous ligand of the cobalt was measured for Co(II)-HCA II in solution containing 0.8 M citrate. Citrate is not an inhibitor of the catalytic activity of Co(II)-HCA II and was not observed in crystal structures. The X-ray structures at 1.5–1.6Å resolution of Co(II)-HCA II were determined for crystals prepared at pH 6.0, 8.5 and 11.0 and revealed no conformational changes of amino-acid side chains as a result of the use of citrate. However, the studies of Co(II)-HCA II did reveal a change in metal coordination from tetrahedral at pH 11 to a coordination consistent with a mixed population of both tetrahedral and penta-coordinate at pH 8.5 to an octahedral geometry characteristic of the oxidized enzyme Co(III)-HCA II at pH 6.0.

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Acid-base properties of cobalt(II)-substituted carbonic anhydrases

Cited by 31 publications

References 0 publications

Mimicking natural evolution in metallo-β-lactamases through second-shell ligand mutations

Mimicking natural evolution in metallo-β-lactamases through second-shell ligand mutations

Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: Spectral evidence for allosteric regulation by pH and bicarbonate ion

Comparison of solution and crystal properties of Co(II)–substituted human carbonic anhydrase II

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